b'Frequently asked questions Below are some common questions and answers to help you start or troubleshoot molecular biology experiments.Sample preparation What are the benefits of using random primers, oligo(dT) primers, Which kit should I use to isolate nucleic acids from my sample? gene-specific primers, or oligo(dT)/random mixed primers in Choosing the right product is fundamental to ensuring proper lysis of cells and tissue,reverse transcription?as well as sufficient yield and quality of isolated nucleic acids. Look to our selectionRandom primers are good to use with degraded RNA, RNA with high secondary guides (see pages 911) to help you decide according to nucleic acid type, samplestructure, nonpolyadenylated RNA, or prokaryotic RNA. source, experimental throughput, and format as well as downstream applications. Oligo(dT) primers are an optimal choice for synthesis of full-length cDNA from eukaryotic mRNA. Applications include cDNA cloning, cDNA library construction, What are the key steps to preventing RNA degradation?and 3 rapid amplification of cDNA ends (3 RACE).The basic lab precautions listed below can help minimize RNA degradation and avoid experimental inconsistency and failure. Gene-specific primers are designed based on known sequences of the target RNA. These primers offer the most specific priming and are commonly used in one-step Use nuclease-free pipette tips and tubes RT-PCR. Use nuclease-free water and reagents A mixture of oligo(dT) and random primers is often used in two-step RT-PCR to achieve the benefits of each primer type (see page 18). Regularly decontaminate work surfacesFor more tips and troubleshooting advice on reverse transcription, visit Properly stabilize RNA sources before storage thermofisher.com/rteducation and thermofisher.com/rtsupport.For more tips and troubleshooting advice on sample prep, visit thermofisher.com/rnabasics and thermofisher.com/napsupport. PCR amplificationHow can I optimize primer annealing for PCR?Reverse transcription Traditionally, gradient thermal cyclers have been used to simultaneously assess How do I improve the efficiency of cDNA synthesis when workinga number of temperatures around the theoretical annealing point. Compared to with challenging samples (e.g., low-abundance, degraded,gradient thermal cyclers, instruments with the VeriFlex technology allow more precise inhibitor-containing, or GC-rich RNA)? temperature control for faster optimization of primer annealing (see page 21).When working with challenging RNA samples, select a reverse transcriptase that isTedious optimization steps may be circumvented using the novel Platinum DNA highly sensitive, processive, thermostable, and resistant to common inhibitors, to helppolymerases. Their innovative buffers enable specific annealing at 60C for you obtain the highest cDNA yield (see page 15). most primers when they are designed following general primer design rules (see pages 2829).ResourcesContents 51'